Osteoarthritis (OA), the most common form of arthritis, is characterized by the destruction of articular cartilage. The main constituents of articular or joint cartilage are type II collagen and various proteoglycans, such as aggrecan, chondroitin sulfate, and hyaluronan. Matrix metalloproteinase 13 (MMP-13) has been shown to be main collagenase responsible for degradation of articular cartilage during OA. Multiple attempts to develop an MMP-13 inhibitor-based drug failed mostly due to the dose limiting side effects collectively known as musculoskeletal syndrome (MSS). While the exact cause of MSS is not known it is believed to be due to the lack of selectivity of drug candidates towards other representatives of metalloproteinase families. Identification of protease secondary binding sites (exosites), i.e. non-active site regions that facilitate or modulate protease activity, could be utilized for the design of selective inhibitors within protease families. Our laboratory has developed triple-helical peptide (THP) fluorescence resonance energy transfer (FRET) substrates for high throughput screening (HTS) of collagenolytic MMPs and identification of putative exosite-binding compounds. Using HTS techniques with FRET THP assays, we previously identified several selective low micromolar inhibitors of MMP-13. One inhibitor (compound Q/4, PubChem CID 2047223) exhibited properties suggesting that its mode of action was not via Zn chelation. Compound Q/4 possessed a significantly smaller molecular scaffold than previously described MMP-13 inhibitors, yet offered excellent selectivity. We wish to further develop compound Q/4 and its analogs as in vivo MMP-13 probes. The specific aims to achieve this goal are as follows: (1) X-ray crystallography-guided medicinal chemistry of MMP-13 exosite probes; and (2) in vitro and in vivo testing of MMP-13 exosite probes. The present work will lead to in vivo probes that can evaluate the effects of selective inhibitors on extracellular proteolytic activity in OA and lay the groundwork for the development of novel MMP imaging agents. In addition, a unique mode of MMP inhibition will be identified.